The aim of tissue engineering is to develop biological substitutes that restore, maintain, or even improve tissue and organ function based on the combined principles of engineering and material and life sciences.¹  The key components involved in engineering tissues are cells, scaffolds, and growth-stimulating signals.²  Early stage tissue engineering scaffolds were made of bio-inert materials with suitable combinations of mechanical properties to adequately meet those of the replaced tissues.²  Thus, the scaffolds elicited minimal responses from host tissues.²  On the other hand, second-generation biomaterials were intended to elicit controlled reactions within the implanted tissues and to be resorbable.²  Furthermore, modern biomaterials of the third generation are being developed to support and stimulate the regeneration of functional multicellular tissues.²  Thus, the development of smart materials is an issue associated with the third generation of biomaterials.

Tissue dynamics, that is, tissue formation, maintenance of structural integrity and homeostasis, repair after injury, and regeneration, are a result of spatiotemporal coordination of cell differentiation, proliferation, and migration (Figure 1.1).³  These cellular processes are regulated by a set of complex and highly dynamic physical and chemical cues in the extracellular environment.^4–7  Recapitulation of dynamic signaling can be achieved by “smart materials”, which are designed with physical and/or chemical properties that significantly change in response to small external stimuli.

Cells in multicellular tissue can sense a set of complex biophysical and biochemical signals and can integrate these inputs to greatly change the dynamic state of the cells. In this context, the cell is itself a “smart material”. The key to fully exploiting the potential of smart materials for tissue scaffolds is to design dynamic physical and chemical cues based on the understanding of the cell as a “smart material”. Specifically, insights regarding the effect of small localized changes in the material properties of the extracellular matrix (ECM), such as local chemical composition, micro-/nanotopography, and stiffness of scaffolds, on cells found in multicellular tissues are of particular importance in the smart design of materials for tissue engineering.

This chapter describes a design concept for smart materials in tissue engineering with a biophysical basis regarding the dynamic and complex interactions of the cell with the extracellular environment. First, Section 1.2 describes the biophysical basis underlying the smart design of materials for tissue engineering, specifically the mechanism through which cells interact with the ECM. Second, in Section 1.3, the determinants of cell fate, e.g., differentiation and the balance between proliferation and apoptosis, and migration in the ECM are discussed. The space and time scales in cell and material interactions are explained in Section 1.4. A phenomenological sense of space and time is important for designing dynamic physical and chemical cues with high efficiency. Finally, in Section 1.5, these insights are summarized into a methodology to design smart materials, which provides dynamic cues for the integrative control of cell differentiation, proliferation, and migration within scaffolds generated by smart tissue engineering.

As mentioned in Section 1.1, insights from studies about cell–ECM interactions and cellular signaling processes are necessary for integration into the smart design of materials for tissue engineering. Figure 1.2 shows the pathway of direct physical transmission and that of indirect mechanochemical signal transduction from the ECM and the nuclear interior. Through this pathway, cells interact with the ECM by sensing material properties, such as the composition of adhesion proteins, micro-/nanotopography, and stiffness of the ECM, and then integrating these inputs, followed by dramatic changes in their dynamic state. The balance of forces between the cells and ECM is affected by changes in the ECM’s material properties, even though these changes are small. Mechanical stimuli from the ECM are transmitted via integrin transmembrane molecules into the cytoplasm (Figure 1.2, right panel), cause changes in the actin organization, and/or are converted into biochemical signals (Figure 1.2, left panel). Both mechanical and biochemical signals finally reach the nucleus, which influence the transcription events in the cell nucleus. Therefore, the signaling from the ECM to the interior of the nucleus through the direct physical link and mechanochemical signal transduction pathway is considered as a basis to plot a strategy for designing smart materials to control cell differentiation, proliferation, and migration for the purposes of tissue engineering.

As illustrated at the bottom of Figure 1.2, extracellular domains of integrin bind to ECM adhesive molecules, such as fibronectin, collagen, and laminin, and in the cytoplasmic region. In the cytoplasm, their intracellular domains bind to integrin-associated proteins, such as talin, paxillin, vinculin, zyxin, and focal adhesion kinase (FAK). This molecular complex physically links the intracellular domains of integrin molecules to the actin filaments, and functions like a molecular clutch to transmit the forces between the ECM and the actin filaments.^8,9  The profile of the assembled proteins specifies the magnitude of the forces transmitted between ECM-bound integrins and actin filaments.^9–11  The profile of the assembled proteins and the rates of association and dissociation¹²  are affected by mechanical force either generated by intracellular actomyosin interactions and/or originating from the external forces applied to the cell. An increase in the forces promotes the assembly of integrin-associated proteins and their stability in the adhesion complex.¹³  The development of focal adhesions facilitates the assembly of actin stress fibers and actomyosin contractile forces.^14,15  On the other hand, a decrease in the actomyosin contractile force results in disassembly of the focal adhesion and actin stress fibers.¹⁶ 

The opposite end of the actin filament links to the chromosomal DNA through nuclear membrane proteins, as illustrated in Figure 1.2 (right panel).^17–20  This physical link functions to transmit the mechanical force directly from the ECM and inner nuclear region, resulting in changes in nuclear morphology^21,22  and the architecture of the nuclear interior, e.g., nuclear matrix distortion, chromatin remodeling, and DNA unzipping.²³  These nuclear architecture changes alter transcriptional activities by affecting the transport of soluble signaling factors and the assembly of transcription factors in the nucleus.^23–25 

The cell–ECM adhesion complex serves not only as a molecular clutch, but also as a node to transduce the mechanical forces acting on the adhesion foci into biochemical signals. The major parts of this signal transduction pathway are shown in Figure 1.2 (left panel). Yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) signaling is known to contribute to the regulation of cell fate, e.g., differentiation in human mesenchymal stem cells (MSCs) and survival in human lung microvascular endothelial cells.²⁶  Additionally, Ras/mitogen-activated protein kinase (MAPK; Ras-Raf-MEK-ERK) signaling^27,28  is known to be involved in the regulation of cell fate, such as proliferation of human MSCs²⁹  and cancer cells,³⁰  and maintenance of keratinocyte stem cells.³¹  The phosphoinositide 3-kinase (PI3K)/serine/threonine-specific protein kinase (Akt) signaling pathway is located downstream of Ras. It is shown that PI3K/Akt signaling functions to regulate the self-renewal of embryonic stem (ES) cells in mice.^32–36 

As explained in Sections 1.2.1 and 1.2.2, the cell adhesion complex and actin cytoskeleton are key players in cell–ECM interactions. Cell adhesion complexes and the actin cytoskeleton reorganize in response to mechanical and chemical inputs from the ECM and regulate intranuclear events by transmitting these signals to the nucleus. Thus, it is predictable that defining the dynamics of cell adhesion complexes and the actin cytoskeleton will lead to control of cell differentiation and proliferation. In addition, the actin cytoskeleton associated with the adhesion complex provides structural support and generates a driving force for cell shape changes and cell migration.³⁷  Therefore, cell differentiation, proliferation, and migration during organ- and tissue-specific differentiation and morphogenesis can be recapitulated by defining the dynamics of cell adhesion complexes and the actin cytoskeleton. In the following section, the mechanism through which adhesion complexes and the actin cytoskeleton regulate cell differentiation, proliferation, and migration is explained.

MSCs have been extensively used to study how cell adhesion complexes and the actin cytoskeleton are associated with the stem cell lineage commitment.^38–40  The osteogenic lineage is typical in cells with mature and elongated integrin clusters and in cells with well-organized actin stress fibers in human, mouse, and rat MSCs (Table 1.1), whereas dispersion of these structures is observed in other lineage types. Adhesion complexes and actin stress fibers are smaller and thinner during commitment of human MSCs to the myogenic, adipogenic, and neurogenic lineages (in order from largest to smallest). In the neurogenic lineage, unidirectional orientation of the cytoskeleton and nuclear elongation lead to upregulation of neuronal lineage commitment in human MSCs.

Studies on stem cell differentiation in relation to adhesion complexes and actin organization in ES cells have continued to advance. As shown in Table 1.1, in human and mouse ES cells, downregulation of integrin clustering and cell spreading upregulates self-renewal of ES cells; these effects result from suppression of extracellular signal-regulated kinase (ERK)/c-Jun N-terminal kinase (JNK) activity through integrin-mediated FAK signaling. In contrast, mesoderm differentiation of mouse ES cells is enhanced due to upregulation of stress fiber formation. In human ES cells, rapid neuronal lineage commitments have been shown to be associated with elongated morphology and cytoskeletal alignment in a single direction.⁴¹  These cell morphological and cytoskeletal changes lead to tensional force transmission to the nucleus and can influence signal transduction and gene expression.⁴¹ 

Studies concerning the regulation of cell proliferation in relation to adhesion complexes and the actin cytoskeleton have been performed on the basis of studies on cell morphology in growth control.^42,43  Generally, cell proliferation is enhanced by cell spreading associated with the maturation of adhesion foci and development of actin stress fibers. The best characterized pathway in this regard is the stimulation of the Rho/Rho-associated protein kinase (ROCK) pathway by increased actin cytoskeletal tension.^28,42,44,45  The increase in the mechanical tension results in promotion of integrin clustering, in turn increasing ERK activity. As a consequence, the cell growth rate is increased through ERK-dependent induction of cyclin D1.

Migrating cells share several basic processes: (i) membrane protrusion at their leading edge, (ii) adhesion to the ECM, and (iii) generation of force against this adhesion to drive forward the cell body.⁴⁶  However, different types of cells adopt different migration modes by modulating actin cytoskeletal organization (e.g., the magnitude of bundling and the polarity), the direction of cell–ECM interactions, and force generation.⁵  Individual cells that have a predominantly cortical actin cytoskeleton and migrate while being loosely contacted to the ECM present rounded shapes and amoeboid type migration, whereas those with highly developed actin stress fibers that migrate while having firm adhesions to the ECM exhibit spindle shapes and develop mesenchymal migration. Cells undergoing mesenchymal migration transiently form and disrupt cell–cell adhesions and are migrating along a structural frame given by the ECM.^47–49  The continuing tight cell–cell contacts can lead to confined migration in each cell inside the multicellular aggregate and yet allow cytoskeletal activity at the outward edge of the aggregate or at the basal cell–substrate contacts. In cases in which the cell aggregate is migrating through a three-dimensional structural frame with a curved boundary, the collective cell migration leads to the formation of a tube or multicellular ball in the hollow (see also Section 1.4.4).^50–52 

As demonstrated in Section 1.2.3, adhesion complexes and the actin cytoskeleton are shared as major components in signaling to regulate cell differentiation, proliferation, and migration, the three key processes for tissue engineering (Figure 1.1). These key processes are regulated by the extracellular environment, which consists of a physical network of proteins and proteoglycans, and also involves nonmatrix soluble components, such as growth factors. All these physical and chemical factors provide a complex material niche and are integrated to regulate multicellular tissue dynamics.^53,54  This section describes specific factors, such as the composition of cell adhesion proteins, ECM topography, and ECM stiffness, that work as local microenvironmental cues and are important for designing smart materials, rather than soluble and highly diffusive factors.

The extracellular microenvironment that surrounds cells is a highly hydrated network that includes molecular signals. These signals include (i) insoluble hydrated macromolecules (fibrillar proteins, such as collagens; noncollagenous glycoproteins, such as elastin, laminin, and fibronectin; and hydrophilic proteoglycans with large glycosaminoglycan side chains), (ii) soluble macromolecules (growth factors, chemokines, and cytokines), and (iii) proteins on the surfaces of neighboring cells.⁷  Within these forms, insoluble hydrated macromolecules as local cues are of primary importance while considering the design of smart materials. The interactions between integrins and these macromolecules are dynamic and spatially precise and can initiate intracellular signaling events for the integrative control of cell differentiation, proliferation, and migration.⁵⁵  Specifically, interactions with fibronectin have been extensively studied and widely utilized to specify cell adhesive patterns on cell and tissue culture substrata.⁵⁶ 

There is a wide variety of ECM topographical features among different tissues and organs and even within the same tissue. In general, tissues are either coarse and soft as in loose connective tissues⁵⁷  and embryo stroma,⁵⁸  or densely packed and stiff as in tightly connective tissues, tumor stroma,⁵⁹  and bones.^60,61  The ECM topography serves as structural restrictions at several sizes (detailed in Section 1.4). These structural restrictions alter the shape of the cell–ECM boundary and affect mechanical homeostasis between the cells and ECM;⁶²  this process leads to reorganization of cell adhesion complexes and the actin cytoskeleton. In this manner, ECM topography affects cell differentiation, proliferation, and migration.

As described in Section 1.3.2, micro-/nanotopography functions as a structural restriction. In addition, tuning the stiffness of the topography allows modulation of the magnitude of the restriction effect. Various cell types, including human MSCs,^63–65  mouse ES cells,⁶⁶  fibroblasts,^67–69  and glioma cells,⁷⁰  have been reported to sense the stiffness of the ECM within the range of in vivo soft tissue stiffness (Figure 1.3, bottom)⁷¹  and change the spatial organization of the actin cytoskeleton and adhesion complex. For the engineering of smart materials, this cellular property can be utilized to externally adjust the strength of the cytoplasm–ECM engagement (Figure 1.2), e.g., the magnitude of the restriction effect of the topography.

The mechanism that modulates the strength of the cytoplasm–ECM engagement is shown in Figure 1.3. In cells on a stiff (>10 kPa) ECM, the rate of association of integrin-associated proteins and their stability in the adhesion complex are increased by mechanical tension,⁷²  which leads to maturation of adhesion foci into focal adhesions. The formation of the matured focal adhesions facilitates the development of prominent actin stress fibers in the cytoplasm.^65,70,73  On the other hand, in cells on a soft ECM (<10 kPa), cell–ECM adhesion foci, which are forced by the actively contracting actomyosin, move with the ECM deformation. The cells on such a compliant ECM respond to maintain a constant ECM deformation by dispersing actin stress fibers and adhesion complexes, thereby decreasing the stress at the adhesion point.⁶⁸  Therefore, substrates with stiffness lower than 10 kPa can be utilized to tune the strength of the cytoplasm–ECM engagement, i.e., the magnitude of the restriction effect of the micro-/nanotopography. This parameter can be adjusted by increasing or decreasing the stiffness of the ECM.

As described in Section 1.3, cells significantly change their dynamic state by sensing the composition of adhesion molecules, the ECM topography, and the ECM stiffness. In addition to the dominant ECM factors, a sense of space and time will be helpful for optimal design of “dynamic” cues. In this section, spatial (Figure 1.4) and temporal (Figure 1.5) scale concepts of the cell–ECM interaction are presented.

As described in Section 1.3.1, binding of an integrin to its ligand on the material surface is an initial point of signaling that regulates cell differentiation, proliferation, and migration. As illustrated in Figure 1.4 (boxed area at the bottom), resting integrins are bent, with the ligand binding site less than 5 nm from the membrane anchor, and activated integrins are in the extended conformation with the ligand binding site 15–20 nm away.^74,75  The lifetime of a bond is estimated at the level of a single molecule.⁷⁶  The measurements show that the lifetime of a bond between an integrin and fibronectin fragment is force dependent, and the force prolongs the bond lifetime in the range of 10–30 pN, as shown in Figure 1.5C, within 10 s.⁷⁶ 

The integrin clustering process is summarized in Figure 1.5B. The initial form of cell attachment to the substrate through integrin binding is nascent adhesions, smaller than the ∼0.25-µm resolution of the light microscope.^15,77  Some of the nascent adhesions disassemble within minutes,^15,77  whereas the others grow and mature into focal complexes (∼0.5 µm) and then into focal adhesions (1–5 µm) within 10 min.⁷⁸  Focal adhesions then either disassemble within 10–20 min^79,80  or further develop into stable fibrillar adhesions (>5 µm) that are involved in ECM remodeling.^15,80 

The size threshold of the integrin cluster to engage actin filaments and thus to form the mechanical link between the ECM and the cytoplasm has been characterized at the single-molecule level by using nanofabricated surfaces.^81–83  A group of integrins spaced at less than 70 nm (Figure 1.6) links the actin filament and transmits force between the ECM and cytoplasm. Three⁸⁴  or four⁸¹  integrin molecules are required to engage actin filaments to transmit the forces between the ECM and cytoplasm.

It is reasonable to assume that the topographical features act directly on cellular factors of the same size. As predicted, topographical features ranging from tens to hundreds of nanometers directly affect the assembly and disassembly of the integrin cluster, such as nascent adhesions (<0.25 µm), focal complexes (∼0.5 µm), and focal adhesions (1–5 µm), as a structural restriction. Representative elements that affect integrin clustering include nanodots, nanopits, and nanogrooves.^4,85–87  In cells cultured on these nanostructured surfaces, the accessibility of the cell membrane is specified by the feature sizes, i.e., the width and height/depth, of the nano-element and by the spacing between these elements. Generally, narrowly spaced elements of more than 40 nm in height/depth can constrain integrin molecules only on the top surface of the topographical elements.^83,85  Specifically, an array of dot structures with a diameter smaller than 100 nm and spaced between 100 nm and 1 µm can effectively influence integrin clustering.⁸³  Integrin clustering is restored on an array of dot structures with too small a spacing (<100 nm) by connection of intracellular integrin-associated proteins with neighboring integrin domains limited on top of the dot structures.⁸³  Additionally, spacing between nanodots of more than 1 µm restores integrin clustering on the bottom surface by increasing the plasma membrane accessibility to the bottom area.⁸⁵  In a manner similar to that of nanodot structures, an array of nanopit structures^85,88–90  and parallel nanogrooved structures^83,91,92  can also affect the organization of integrins. In this way, nanotopographical features modulate the spatial distribution of cell adhesion complexes and the associated actin filaments, thus affecting the regulation of cell differentiation, proliferation, and migration.

Feature lengths of actin filaments range from submicrometers to 10 µm. Thus, topographical features ranging from submicrometers to 10 µm in size are thought to directly affect actin filaments as structural restrictions. Actin reorganization guided by topographical features at this scale has long been studied as “contact guidance”.^4,93–96  Based on the concept of contact guidance, mechanical restriction of the cytoskeleton lowers the probability of traversing obstacles, such as dots and ridges, by actin filaments. Simple synthetic microtopographical surfaces, such as micropillar arrays, parallel ridges, and grooves, have been used to characterize contact guidance.

Consistent with the idea of contact guidance, an array of narrowly spaced micropillars steers the bundled actin filaments, limiting contacts only to the top of the pillars,^97–99  as illustrated in Figure 1.7A. In contrast, if there is sufficient spacing between the pillars, as shown in Figure 1.7B, bundled actin filaments are guided to form between the pillars.⁹⁸  The threshold of spacing that defines localization of actin filaments is 10 µm in 3T3 fibroblasts⁹⁸  (Figure 1.7). The threshold of spacing differs among cell types due to differences in the organization and resulting flexibility of the actin cytoskeleton.^96,100  The pillar diameter threshold that specifies whether the actomyosin contractile fibers form along the edge of the pillar or between neighboring pillars is 1 µm, which has close correlation to the size of a unit of the actomyosin fiber (boxed area in the middle in Figure 1.4).^101,102 

At the single-cell level with a feature length of 10–100 µm (top panel in Figure 1.4), fibronectin micropatterns can control the cell spreading area, cell shape, and tension in the actin cytoskeleton. As explained in Section 1.2.3, these dynamic changes in the actin cytoskeleton significantly affect cell differentiation, proliferation, and migration. The feature timescale of these cellular processes varies from hours to weeks, as summarized in Figure 1.5A.

The feature time of cell differentiation ranges from days to several weeks. For example, human MSCs differentiate into osteogenic, chondrogenic, myogenic, adipogenic, and neurogenic cells typically in several weeks.^65,103–105  Human fetal pancreatic development occurs over weeks, whereas directed differentiation in vitro is measured in days.¹⁰⁶ 

In terms of the timescale of cell proliferation (Figure 1.5A), the overall cell cycle of the human ES cell is 15–16 h, which is shorter than the typical 24-h cell cycle of differentiated somatic cells.¹⁰⁷  The pluripotency of human ES cells in vivo is transient, and their cell cycle duration becomes more than 24 h.¹⁰⁸  The cell cycle of human iPS cells is 40–50 h¹⁰⁹  or less (about 24 h),¹¹⁰  depending on the culture conditions.

The timescale of cell migration (Figure 1.5A) depends on the modes of migration.⁵  Fibroblasts, which have well-bundled actin stress fibers that are tightly attached to the ECM via focal adhesions, are spindle-shaped and exhibit mesenchymal migration with a speed of 0.1–1 µm min⁻¹.⁵  In contrast, cells that are loosely attached to the ECM migrate faster.⁴⁶  Specifically, leukocytes can move virtually within any substrate found in the body at speeds up to 100 times faster than mesenchymal cells, in order to achieve an effective immune response.

A feature length of more than 100 µm corresponds to the size of multiple-cell aggregates. As illustrated in Figure 1.8A, cells on conventional rigid plastic or glass planar surfaces, i.e., two-dimensional (2D) cultures, are steered to develop into actin stress fibers that associate with the mature adhesion foci on the basal area. Cells under this type of restriction sense force as stresses parallel to the planar basal surface and thus are confined to the 2D surface, forming a planar interface with the surface. Cells on mineralized bone or teeth and a 2D basement membrane layer are under the same restriction.^111,112 

When the 2D surface is modified with fibronectin patterns of 100 µm or more, the patterns contribute to the formation of gradients of mechanical stresses generated within the multicellular aggregates.¹¹³  The patterns of cell differentiation,¹¹⁴  proliferation,¹¹³  and migration^115,116  in the multicellular aggregate can be controlled depending on the mechanical stress induced by the fibronectin patterns.

In three-dimensional (3D) culture conditions, e.g., in the coarse fibrillar 3D network typically observed in the majority of the connective tissues,^57,117  cells can penetrate into the network. Under this type of restriction, cells disassemble the stress fibers and adhesion foci¹¹⁸  in order to maintain mechanical homeostasis with an environment consisting of curved surface areas (Figure 1.8B).⁶² 

As outlined in Sections 1.3 and 1.4, the composition of cell adhesion proteins (Section 1.3.1), ECM topography (Section 1.3.2), and stiffness (Section 1.3.3) act on the integrin molecules (Section 1.4.1), the molecular assembly of integrins and actin (Section 1.4.2), single cells (Section 1.4.3), and multiple-cell aggregates (Section 1.4.4). These multiple spatiotemporal effects are integrated through downward and upward causation to the control of cellular processes.¹¹⁹ 

Scaffolds with a hierarchical structure can have effects resulting in a more sophisticated modulation of cellular processes. For example, nanotopographical cues incorporated into the microstructure have the advantages of both nanotopography and microtopography. Indeed, usefulness of hierarchical structures has been shown to enhance MSC adhesion and proliferation through scaffolds consisting of microscale strands with micro-/nanosized fibers deposited on the surface.¹²⁰  Microspheres coated with nanowires exhibit excellent in vivo biocompatibility while retaining high loading capacity for various cell types.¹²¹  When designing materials for which multiple parameters can be tuned, the integrative effects originating from multiple material properties should be carefully investigated.

Figure 1.9 shows a proposed methodology to design smart materials for tissue engineering to give dynamic local cues for controlling tissue formation, maintenance of integrity and homeostasis, repair after injury, and regeneration. As explained in Section 1.1, these tissue dynamics result from spatiotemporal coordination of cell differentiation, proliferation, and migration. In Section 1.2, the review of the regulatory mechanisms of cell differentiation, proliferation, and migration has demonstrated that these cellular processes are regulated through signaling pathways from the ECM to the nucleus (Section 1.2.1 and 1.2.2) via the cell adhesion complex and actin cytoskeleton. Based on this insight, the design procedure of smart materials for exogenous control of tissue dynamics may be separated by two steps mediated by modulation of cell adhesion complexes and the actin cytoskeleton.

As shown in Figure 1.9A, the first step is to clarify a customized strategy to modulate cell adhesion complexes and the actin cytoskeleton to control cell differentiation, proliferation, and migration for each targeting medical application (the upper boxed area). As demonstrated in Section 1.2.3, a shared common basis for controlling stem cell differentiation (Section 1.2.3.1), proliferation (Section 1.2.3.2), and migration (Section 1.2.3.3) is to modulate the degree of bundling and orientation of actin stress fibers and to regulate the development of adhesion complexes.

Once it is clarified how the adhesion complexes and actin cytoskeleton are controlled for modulation of cell differentiation, proliferation, and migration, the second step is to design the dynamic architecture of a smart material to control the dynamics of the adhesion complexes and the actin cytoskeleton. A key for designing the dynamic architecture is to extract the dominant factors from a limited set of manageable parameters.⁵⁵  Three of the key factors are introduced in Section 1.3 and include (i) composition of cell adhesion proteins, (ii) ECM topography, and (iii) ECM stiffness, which work as the effective local microenvironmental cues. As described in Section 1.4, the sense of the spatial and temporal scale in the interaction between these dominant factors and the relevant cellular factors is important in considering how to build these factors into smart materials. More sophisticated spatial and temporal control of adhesion complexes and the actin cytoskeleton will be achieved by utilization of not only one factor but also a combination of multiple factors. For example, as illustrated in Figure 1.9B, micro-/nanotopographical features function as structural restrictions (Section 1.3.2) at multiple scales (Section 1.4), and the magnitude of the restriction effect can be defined by its stiffness (Section 1.3.3).

This methodology based on two independent steps can be used to overcome the issues of complexity and diversity caused by a variety of requirements of medical applications. It significantly narrows the search to the design variable and generates smart materials with no comprehensive screening but based on the knowledge of the mechanics of how adhesion complexes and the actin cytoskeleton contribute to the regulation of the desired cellular processes. The designed dynamic cues have the advantage of stable control with fine spatial resolution. Again, in tissue engineering, the key cellular processes controlled by the guiding cues are stem cell differentiation, the balance between proliferation and apoptosis, and cell migration. These cellular processes are influenced not only by whether the guiding cues are present, but also by their absolute and relative amounts, their spatial arrangements with molecular and subcellular resolution, and their spatiotemporally dynamic properties.

Another advantage of dynamic cues designed based on the two-step strategy is that non-biomimetic cues affecting cellular processes beyond the effects of the stimuli physiologically provided in vivo may be engineered.³  This effect of synthetic cues can be yielded through drawing together of the cell biophysical insights collected in step 1 of the concept shown in Figure 1.9 and the refinement of the methodology while designing the smart materials for external regulation of cell adhesion complexes and the actin cytoskeleton in step 2.

The key to uncovering the potential of smart materials is a fundamental understanding of the spatiotemporally dynamic interaction between “smart” cells and materials and the mathematical formulation of the current qualitative characterization.³  Further challenges to utilize smart materials for tissue engineering arise in the application of dynamic local cues synergistically with soluble and highly diffusive factors for systemic and global control.

This work was partially supported by the Advanced Research & Development Program for Medical Innovation from the Japan Agency for Medical Research and Development, AMED.